Method and apparatus for optical interactance and transmittance measurements

ABSTRACT

A method, system and apparatus for improving at least one of a) optical interactance measurements, b) optical transmittance measurements and c) optical reflectance measurements. The apparatus has a probe having a body portion and an tip portion. The body portion has a central tubular element having an opening therethrough. The tip portion has a central aperture and a number that is a plurality of ring openings therein. At least some of the plurality of rings are angled with respect to a longitudinal axis of the probe. A plurality of fiber optic bundles corresponds in number at least to the number of ring openings, and at least one fiber optic bundle is arranged at one end of the longitudinal axis for receiving specimen information and at another end of the longitudinal axis connected to a detector for receiving a signal.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/818,289 filed Mar. 14, 1997, which is a continuation of Ser. No.08/385,073 filed Feb. 7, 1995, which is a continuation of Ser. No.08/062,738 filed May 14, 1993, which is a continuation of 07/663,144filed Mar. 1, 1991.

FIELD OF THE PRESENT INVENTION

The present invention relates to an improved method and apparatus forperforming optical interactance and transmittance measurements and, inparticular, such method and apparatus where undesired information isdiscriminated against and desired information is enhanced. Reflectancemeasurements on small amounts of specimen are also encompassed by theinvention.

BACKGROUND OF THE PROBLEM SOLVED BY THE PRESENT INVENTION

When optical energy is transmitted through a diffuse medium, scatteringcauses redirection of the rays so that the geometric pathlength betweenthe energy entrance point and the energy exit point no longer definesthe distance energy travels within the specimen. In addition,substantial energy may be scattered back towards the entrance orotherwise away from the exit region where detection occurs, therebyreducing the detected signal. This signal is therefore variabledepending on the scattering characteristics of the particular region ofthe specimen traversed by the optical radiation.

A further cause of interference is nonhomogeneous or layereddistribution of specimen characteristics, e.g., the layers of skin andfat which cover muscle tissue, the skin which covers the flesh of afruit or vegetable, or the coating of windows through which measurementsare to be made. Often, it is desirable to eliminate the effects of thesurface layers to provide information on the underlying portions of thespecimen. The present invention is directed to solving these problemswhich cause inaccuracies in spectroscopic determination of qualitativeor quantitative characteristics of the specimen.

An additional problem is the making of diffuse reflectance andtransmittance measurements on small specimens. Present reflectanceinstruments are generally designed to illuminate the specimen and detectreflected energy over several square centimeters of area. It issometimes necessary to work with small amounts of specimen, for examplea single seed, which must be recovered intact for future use. Thepresent invention also addresses both diffuse transmission and diffusereflection measurements of small specimens.

BACKGROUND PRIOR ART

There has been a proposal for use of a transmission cell which had twodifferent pathlengths through the specimen as a means of extending thedynamic range of spectral measurements in clear solutions. This proposaldid not encompass separate measurement of the signals for the twopathlengths but rather the combined optical energy was detected. Thisresults in a very nonlinear signal relative to concentration of ananalyte within the specimen, however, the nonlinearity is predictablebased on the known optical geometry of the cell.

Dual pathlength transmission cells with separate detection have beenproposed to remove the effects of window coating in transmissionmeasurements through clear liquids. This approach is equivalent toplacing a second cell of different thickness in the reference beam of adual-beam spectrometer. Effects common to the two paths, such asabsorption due to the window material, equal deposits on the windows,atmospheric absorption, and the specimen absorption in the equal portionof the pathlength are canceled by taking the simple ratio of the signalsderived from the two paths.

Norris [“A New Approach for the Estimation of Body Composition: InfraredInteractance”, Joan M. Conway, Karl H. Norris and C. E. Bodwell,American Journal of Clinical Nutrition, Vol. 40, pp. 1123-1130 (1984)]first proposed measurement by means of “interaction”, whereby a diffusespecimen is illuminated at one location and energy is collected somedistance away on the same surface of the specimen. This is similar todiffuse reflection in that the primary mechanism returning energy to thedetector is scattering, i.e., in the absence of scattering within thespecimen, the incident energy would not impinge on the detection region.It differs from diffuse reflection, however, because the detectionregion does not include the illumination region, but is separated fromit by some distance. Therefore, surface reflection of energy does notcontribute to the detected signal and all the detected energy hastraversed a minimum distance within the specimen, the separationdistance between the source and detector. In this sense, “interaction”is similar to diffuse transmission. In general, the effective depth ofpenetration and the effective pathlength both increase as the spacingbetween the source and detection locations is increased.

Norris and others applying his method have used a single measurement ofthe energy passing through the specimen from the source region to thedetection region. Typically, uniform geometric spacing between thesource area and the detection area is provided by using a centralaperture surrounded a small distance away by a ring aperture. Eitheraperture could serve for the source while the other is used fordetection. Both apertures are usually in contact with the specimen toprevent energy from leaking between the source and detection regionswithout traversing the specimen although thin windows between theapertures and the source have been used. An alternative structure hasbeen to use equally parallel slit apertures, alternating between sourceand detection functions. In this case, all the source slit apertureswere illuminated through one fiber optic bundle while energy wascollected from all the detection apertures by means of a second bundle.Therefore, although more than two apertures exist, there is only onedetected signal.

Diffuse transmittance and reflectance measurements are usually made onlarge volumes of specimen to reduce errors by averaging theinhomogeneities. When only small specimens are available, the usualprocedure is to grind each specimen into a fine powder and mix it with anonabsorbing diluent or to spread it on a diffusely reflectingbackground so as to present a large area for reflectance measurement.There has been a proposed use of a reflecting cone into which thespecimen is placed. The incident energy which does not impinge thespecimen is returned in the direction of the source, and is rejected bythe diffuse reflectance detection geometry. Norris [“Determination ofMoisture in Corn Kernels by Near-Infrared Transmittance Measurement”, E.E. Finney, Jr. and Karl H. Norris, Transactions American Society ofAgricultural Engineers, Vol. 21, pp. 581-584 (1978)] has made diffusetransmission measurements on single seeds by focusing the energy on theseed and placing a large area detector behind the opposite side. Carefulattention must be paid to block the direct path past the seed. Thesemethods for handling intact small samples have been inconvenient atbest.

SUMMARY OF THE INVENTION

In accordance with the invention, a method for improving opticalinteractance measurements comprises the steps of providing illuminationby way of a plurality of paths through a specimen having acharacteristic to be measured, sensing two or more independent signalsdeveloped at the same time or in rapid sequence representing opticalinformation from said specimen and processing said signals in accordancewith appropriate modeling techniques to minimize inaccuracies inspectroscopic determination of qualitative of quantitativecharacteristics of the specimen.

Also in accordance with the invention, apparatus for improving opticalinteractance measurements comprises means for providing illuminationthrough a specimen having a characteristic to be measured along aplurality of different paths, means for sensing optical informationprovided from an illuminated specimen, means for developing a pluralityof independent signals corresponding in number to said plurality ofpaths, the signals representing the optical information obtained fromthe specimen and means for processing the signals in accordance withappropriate modeling techniques to minimize inaccuracies inspectroscopic determination of quantitative or qualitativecharacteristics of the specimen.

For a better understanding of the present invention, reference is madeto the following description and accompanying drawings while the scopeof the invention will be pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings,

FIG. 1 is a cross-sectional, length-wise view in partially schematicform of a two-ring, central aperture probe in accordance with theinvention; and

FIG. 2 is a diametric cross-section of a probe exhibiting a multiplering aperture structure in accordance with the invention.

DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

A first aspect of this invention comprises the use of three or moreoptical apertures shared among the source and illumination functions soas to provide two or more independent signals for further processing andanalysis. For example, in FIG. 1, a probe utilizing fiber optics isshown which has two ring apertures surrounding a central round aperture.

In FIG. 1, the probe 10 includes a cylindrical outer body 30 in which aconcentric inner body 31 is arranged. The inner body 31 tapers at theexamining end to a central aperture 15. The outer body 30 constricts atthe examining end to define a conical, inwardly directed wall. Thetapered end of the inner body 31 defines a second conical wall. Betweenthese two walls 30, 31 is disposed a conical dividing element 33. Theangles defined by the two walls and the conical dividing element arepreferably the same. Conical annular spaces or rings (shown incross-section as 14, 13, 12 and 11) are defined by the two walls anddividing element. In these annular spaces or rings are disposed, inconical fashion, optical fibers 16 and 17 for supplying illumination tothe specimen. Within the inner body 31 are disposed one or more lenses20 for focusing and transmitting incident light entering the aperture15. A central fiber element 21, supported by element 32, receives lightfrom the lenses 20, and directs it to the exit portion 21′ of the probe.Illumination for fibers 16 and 17 is provided at 16′ and 17′.

In this apparatus, each ring is used for illumination by fiber opticelements while the central detection aperture is connected by fiberoptics to a detection system, such as a diode-array spectrophotometer.The central aperture in one embodiment is 1.2 mm diameter, and the innerring has a mean diameter of 6 mm with a width of 1 mm. The mean spacingfrom the inner ring to the central aperture is therefore 3 mm and theminimum spacing is 1.9 mm. The outer ring has a mean diameter of 12 mmand a width of 1 mm, providing a mean spacing from source to detectionof 6 mm and a minimum spacing of 4.9 mm.

In a preferred arrangement, the tip portion of the probe and the fiberoptic elements at the tip portion are angled at approximately 26° withrespect to the longitudinal axis of the probe.

It will be noted in FIG. 1 that the central detection assembly ismovable axially. For interactance measurements, the detection apertureat the distal end of this assembly is normally positioned in the sameplane as the source ring apertures, however, it may be moved back andlenses inserted so as to image a detection area on the specimen into theaperture of the detection fibers. The lenses 20 in the inner body of theprobe are interchangeable and the positions of the lenses and thecentral detection assembly are adjustable by means of spacers. In oneembodiment, lenses and arrangements for three different specimen sizesfor reflectance and one arrangement for interactance are listed in thefollowing Table 1: TABLE 1 REFLECTANCE REFLECTANCE REFLECTANCE StackSpecimen Size Position 1.2 mm 0.7 mm 2.0 mm INTERACTANCE 1 21 mm flength 21 mm f length 15.9 mm spacer Fiber Optic Holder Plano-ConvexPlano-Convex for central fiber Melles Griot Melles Griot 01LPX02301LPX023 2 6 mm spacer 6 mm spacer Melles Griot Spacer 01LPX065 3 21 mmMelles 36 mm Melles 6 mm spacer Spacer Griot 01LPX023 Griot 01LPX065 417.7 mm spacer 17.7 mm spacer 21 mm Melles Spacer Griot 01LPX023 5 15 mmspacer 15.9 mm spacer 17.7 mm spacer Spacer 6 Fiber Optic 15 mm spacer15 mm spacer Spacer Holder for Central Fiber 7 15.9 mm spacer FiberOptic Fiber Optic Spacer Holder for Holder for Central Fiber CentralFiber

This reimaging allows control of the size of the detection area and theangular cone within which energy is detected by changing the lenses andtheir spacing.

It may also be seen in FIG. 1 that the energy source fibers are arrangedin conical form. The energy exits at a mean angle of approximately 45°based on the angle of the fibers and the refraction at their polishedends. For interactance measurements on specimens which have limitedbackscatter, introduction of the energy at an angle directed toward thedetector improves the efficiency. This feature also provides for diffusereflectance and transmittance measurements using the same probe asdiscussed below.

In order to obtain separate signals for each spacing, the two sourcerings may be alternately illuminated or the source energy may bemodulated differently, e.g., at two different frequencies or withdifferent time sequence codes. The detection signal is then gated ordemodulated to separate the information from the two different sources.Each source fiber optic bundle, preferably, has a small percentage ofits input fibers brought out so that the associated source intensity andmodulation can be monitored. It will be obvious to one skilled in theart that additional source rings may be provided, each with itsdistinctive modulation, and that the operation may be reversed toprovide a single source by using two or more detection rings coupled tomultiple or time-shared detection means. For example, FIG. 2 shows adesign comprising 10 large area (8 to 28 mm² active area) ringapertures, nominally designated as source rings, and a central apertureplus 2 additional rings of smaller area (2.9 to 3.4 mm²) nominallyconsidered as detection rings. The source ring active area is adjustedby the density of active fibers within each ring. This structure allowsselection of a wide variety of spacings and locations for themeasurements using combinations of the three different detectionapertures and 10 source apertures. While the examples shown here showring geometries, other geometries, such as parallel slits or smallapertures, which provide substantially constant values of the spacingbetween all points within a given source aperture and those within agiven detection aperture may be used.

Having derived separate signals for the two or more paths, they areprocessed and combined in accordance with a linear or nonlinear model ofthe system response to variations in the concentration of the analytesand interferences present in the spectrum. In the simplest cases, forexample, the cancellation of the optical effects of deposits on thewindow through which the measurements are being made, it may suffice touse the ratio of the signal from one spacing to that of a secondspacing. This assumes that the deposits have the same transmissionspectrum T₁ for both paths, as would be true for a uniform coating, andthat the specimen behind the window is relatively homogeneous with aninteractance spectrum, I. The signals may then be expressed as K₁*T₁*I₁and K₂*T₁*I₂ where K₁ and K₂ are system functions involving the relativesource intensities, the gain through the system, scattering losses andsimilar factors. The ratio is therefore (K₁/K₂)* (I₁/I₂) and the windowcoating transmission has been eliminated from the result. Note that anyfactors common to K₁ and K₂ are also canceled as in the normal use of areference and the remainder factors may be adjusted so that the K factorbecomes unity. Taking the log of the ratio yields “absorbance” A equalto log(I₁)−log(I₂). If I is exponentially related to the product spacingt and the analyte absorptivity a, log(I) will equal (a*t) and the logdifference becomes a* (t₁−t₂) where a is the “absorptivity” spectrumwhich is linearly related to concentration. In many, if not most,practical cases, log (I) is not linearly related to the product ofspacing with absorptivity and absorptivity is not linearly related toconcentration. Therefore, this invention contemplates use of otherlinear and nonlinear chemometric models to define the relationships andprovide quantitive analyte information.

The situation is further complicated if the specimen is nonhomogeneous,such as the cases with layers described above. Here, the various signalsare derived by absorption of light through different combinations ofmaterials within the specimen. All the signals contain information onthe surface layers while the signals derived from the larger spacingscontain information on the deeper layers that is diminished or lackingin the signals measured with smaller spacings. When it is desired todifferentiate between the information derived from deep within thesample and that obtained from layers closer to the surface, thesesignals may be combined in a linear or nonlinear chemometric model so asto extract the desired information. In this case, it is helpful to havethe input energy for each source aperture as an additional measuredquantity for use in the modeling. Each class of specimens requires adifferent form of model, and subclasses require determination of variousmodel parameters during the calibration process.

Another aspect of the invention is the use of the probe for diffusereflection measurements of small specimens. The specimen may be held ina small hole drilled in a flat plate mounted approximately 2 to 4 mmfrom the end of the fiber-optic probe. It is illuminated via one or bothof the outer ring bundles at an angle of incidence of approximately 45degrees. The plate is finished with a mirror surface so that incidentenergy outside the area of the specimen is reflected at approximately 45degrees from the normal. The diffusely reflected energy is collected byimaging the specimen surface on the central fiber bundle via lenseswhose optical axes are coincident with the axis of the probe assembly.The power and spacing of the lenses may be selected so as to select thedesired sample area. Because the collection is normal to the specimenand specimen holder and energy is reflected by the holder and by anywindow above the specimen at an angle, this specularly reflected energyis rejected from the measurement. If desired, a diffuse or specularreflector may be placed behind the specimen to increase its apparentdepth by a factor of at least two.

Alternatively, the axis of the probe may be positioned vertically withthe apertures at the top and a transparent window, such as a microscopeslide, positioned with its upper surface at the appropriate distancefrom the apertures. Specimens may be placed on the window formeasurement. Three measurements may be made:

-   -   a) no specimen (just a slide);    -   b) specimen    -   c) reference spectrum using a diffuse reflecting material such        as SPECTRALON™ (a trademark of Labsphere, Inc.).

The “no specimen” energy spectrum is subtracted from the spectrum of thespecimen and the spectrum of the reference to correct for residualenergy reflected or scattered from the window.

Still another aspect of the invention is the measurement of diffusetransmission through small specimens. The specimen is mounted in a holeon a plate and illuminated as previously described for reflectancemeasurements. The receiving fiber-optic bundle is placed behind thesample so as to collect the transmitted energy. The conical illuminationpattern is helpful in achieving rapid diffusion of energy within thespecimen thereby improving the repeatability of measurements on smallspecimens. In addition, by using two collecting fiber-optic bundles,simultaneous or time shared measurement of both, the diffuselytransmitted and diffusely reflected energy is possible. The combinationof these two measurements allows additional information to be obtainedconcerning the absorption and scattering characteristics of thespecimen.

Still another embodiment utilizes the probe as described above forreflection and an additional fiber bundle is employed behind thespecimen to illuminate it. Therefore, the same receiver is utilized fortransmittance and reflectance with two different illumination sourcesbeing provided. A switching arrangement may be used to alternate betweenthe illumination sources.

In each case, the central detection element may comprise the detectoritself rather than the fiberoptic detector bundle.

In FIG. 2, thirteen concentric circular regions (A1, A2, A3, a, b, C, d,e, f, g, h, i, and j) are shown. Regions A1, A2, and A3 are detectionapertures and regions a through j are source apertures. The dimensionsand properties of these regions are shown in Tables 2 and 3. TABLE 2Detection Aperture Dimensions Detection Outside Diameter Inside DiameterArea Aperture mm mm mm² A1 1.92 0.00 2.90 A2 7.81 7.55 3.20 A3 15.3115.17 3.37

TABLE 3 Source Aperture Configurations Active Inside Outside Area ActiveArea Ref. Ref. Fiber Region Diameter_mm Diameter_mm mm² Area % mm² Fiber% mm² a 1.92 3.85 8.8 90 7.90 10 0.9 b 3.85 5.70 14.5 90 13 10 1.45 c5.70 7.55 20.2 90 17.2 10 2.0 d 7.81 10.21 34 50 17 5 1.7 e 10.21 12.6643 50 21.5 5 2.2 f 12.66 15.17 52 50 26 5 2.6 g 15.31 17.30 51.1 40 20.44 2 h 17.30 19.30 57.5 40 23 4 2.3 i 19.30 21.30 63.7 40 25.5 4 2.6 j21.30 23.30 70 40 28 4 2.8

While the above described probe is preferred, an alternate embodimentcan be constructed where the direction of light flow can use the lightsource being provided at the control aperture and one or more of thefiber optic bundles responsive to specimen information.

While the foregoing description and drawings represent the preferredembodiments of the present invention, it will be obvious to thoseskilled in the art that various changes and modifications may be madetherein without departing from the true spirit and scope of the presentinvention.

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 27. Apparatus for improving at least oneof a) optical interactance measurements, b) optical transmittancemeasurements and c) optical reflectance measurements comprising: a probehaving a body portion and an tip portion; the body portion comprising acentral tubular element having an opening therethrough; the tip portionhaving a central aperture and a number that is a plurality of ringopenings therein; at least some of the plurality of rings being angledwith respect to a longitudinal axis of the probe; a plurality of fiberoptic bundles corresponding in number at least to the number of ringopenings, and at least one fiber optic bundle arranged at one end of thelongitudinal axis for receiving specimen information and at another endof the longitudinal axis connected to a detector for receiving a signal.28. A method for receiving specimen information and analyzing specimeninformation comprising placing said one end of the apparatus of claim 27against a surface providing specimen information, detecting transmittedspecimen information at the another end, and analyzing detected specimeninformation.
 29. A system for for improving at least one of a) opticalinteractance measurements, b) optical transmittance measurements and c)optical reflectance measurements comprising: the apparatus of claim 27;a processor receiving signals from the apparatus comprising signalinformation, and software in the processor performing an analyticprocedure on the information to provide information regarding thespecimen.